Reanne Bowlby

Recurrent DGCR8, DROSHA, and SIX Homeodomain Mutations in Favorable Histology Wilms Tumors

We report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates that tumors with SIX1/2 and/or miRNAPG mutations show a pre-induction metanephric mesenchyme gene expression pattern and are significantly associated with both perilobar nephrogenic rests and 11p15 imprinting aberrations. Significantly decreased expression of mature Let-7a and the miR-200 family (responsible for mesenchymal-to-epithelial transition) in miRNAPG mutant tumors is associated with an undifferentiated blastemal histology. The combination of SIX and miRNAPG mutations in the same tumor is associated with evidence of RAS activation and a higher rate of relapse and death.

Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles

Summary Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance.

miR-509-3p is clinically significant and strongly attenuates cellular migration and multi-cellular spheroids in ovarian cancer

Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E–3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E–3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3ps most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.

Indoleamine 2,3-Dioxygenase 1, Increased in Human Gastric Pre-Neoplasia, Promotes Inflammation and Metaplasia in Mice and Is Associated With Type II Hypersensitivity/Autoimmunity

BACKGROUND & AIMS Chronic gastrointestinal inflammation increases the risk of cancer by mechanisms that are not well understood. Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-binding enzyme that regulates the immune response via catabolization and regulation of tryptophan availability for immune cell uptake. IDO1 expression is increased during the transition from chronic inflammation to gastric metaplasia. We investigated whether IDO1 contributes to the inflammatory response that mediates loss of parietal cells leading to metaplasia. METHODS Chronic gastric inflammation was induced in Ido1-/- and CB57BL/6 (control) mice by gavage with Helicobacter felis or overexpression of interferon gamma in gastric parietal cells. We also performed studies in Jh-/- mice, which are devoid of B cells. Gastric tissues were collected and analyzed by flow cytometry, immunostaining, and real-time quantitative polymerase chain reaction. Plasma samples were analyzed by enzyme-linked immunosorbent assay. Gastric tissues were obtained from 20 patients with gastric metaplasia and 20 patients without gastric metaplasia (controls) and analyzed by real-time quantitative polymerase chain reaction; gastric tissue arrays were analyzed by immunohistochemistry. We collected genetic information on gastric cancers from The Cancer Genome Atlas database. RESULTS H felis gavage induced significantly lower levels of pseudopyloric metaplasia in Ido1-/- mice, which had lower frequencies of gastric B cells, than in control mice. Blood plasma from H felis-infected control mice had increased levels of autoantibodies against parietal cells, compared to uninfected control mice, but this increase was lower in Ido1-/- mice. Chronically inflamed stomachs of Ido1-/- mice had significantly lower frequencies of natural killer cells in contact with parietal cells, compared with stomachs of control mice. Jh-/- mice had lower levels of pseudopyloric metaplasia than control mice in response to H felis infection. Human gastric pre-neoplasia and carcinoma specimens had increased levels of IDO1 messenger RNA compared with control gastric tissues, and IDO1 protein colocalized with B cells. Co-clustering of IDO1 messenger RNA with B-cell markers was corroborated by The Cancer Genome Atlas database. CONCLUSIONS IDO1 mediates gastric metaplasia by regulating the B-cell compartment. This process appears to be associated with type II hypersensitivity/autoimmunity. The role of autoimmunity in the progression of pseudopyloric metaplasia warrants further investigation.

Abstract S2-04: Comprehensive molecular characterization of invasive lobular breast tumors

Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer accounting for 10-15% of invasive breast tumors. ILC is typically ER+ and beyond the known mutation and/or loss of E-cadherin function, which contributes to a highly discohesive morphology, little is known about the additional mechanisms driving ILC tumorigenesis, or alterations that differentiate ILC from invasive ductal carcinomas (IDC). Methods A dataset of 817 breast tumors from the TCGA Project, including 490 IDC, 127 ILC and 88 samples with a mixed IDC-ILC histology, were profiled on six genomic platforms to develop a comprehensive atlas of mutational, epigenetic, transcriptional and proteomic data. Integrative genomic analyses, both supervised and unsupervised, of ILC tumors and across histological subtypes were performed to identify genomic drivers of ILC oncogenesis. Results Comprehensive multi-platform analyses identified distinct molecular events associated with ILC tumors. As expected, lack of E-cadherin protein, as determined by Reverse Phase Protein Array (RPPA), and CDH1 mRNA expression was uniformly observed in ILC cases associated with distinct alterations targeting CDH1. In addition to previously reported CDH1 and PIK3CA mutations, we identified a number of novel ILC-enriched recurrent mutations targeting PTEN, RUNX1, TBX3, and FOXA1. An increased incidence of PTEN inactivating events, both mutations and copy number changes, were identified in ILC (13%) compared to IDC ER+ (7%), which corresponded with altered PTEN protein expression. These alterations were largely mutually exclusive with PIK3CA mutations and correlate with increased Akt activation as evident by increased Akt phosphorylation (pS473 and pT308), thus identifying a potential therapeutic opportunity for ILC patients. GATA3 signaling, which regulates epithelial cell differentiation, is frequently altered in luminal/ER+ breast cancers. Our analyses determined GATA3 mutations are more frequent in IDC luminal tumors as compared to ILC (19 % vs 5%). ILC luminal tumors show significantly lower GATA3 protein expression, but a higher frequency of mutations in FOXA1 (9% vs 2% in Luminal IDC), a transcription factor required to promote ER transcriptional programs. Within ILC tumors, FOXA1 mutations were found to cluster into a specific region of the Forkhead (FK) DNA binding domain. A broader analysis of FOXA1 mutations in breast and prostate cancer confirm two specific hotspots in the FK domain and the C-terminal transactivation domain. Interestingly, these mutational classes are associated with distinct transcriptional changes suggesting different functional effects. Finally, mRNA-seq analyses identified three robust molecular subclasses that are characterized by distinct genetic, genomic and proteomic patterns, including an increased immune-related group (Class 2), as well as differences in prognosis. Conclusions In this study, we developed a comprehensive atlas of genomic alterations that reveals key molecular differences differentiating ILC (FOXA1) from IDC (GATA3) tumorigenesis, a potential therapeutic target for ILC (Akt), and novel ILC subclasses based on underlying biological events. These findings provide further insight into the molecular heterogeneity of ER+ breast cancer. Citation Format: Giovanni Ciriello, Michael L Gatza, Katherine A Hoadley, Hailei Zhang, Suhn K Rhie, Reanne Bowlby, Matthew D Wilkerson, Cyriac Kandoth, Michael McLellan, Andrew Cherniack, Peter W Laird, Chris Sander, Tari A King, Charles M Perou. Comprehensive molecular characterization of invasive lobular breast tumors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S2-04.

Abstract 4621: Validation and calibration of next-generation sequencing to identify Epstein-Barr Virus-positive gastric cancer

Background: Gastric adenocarcinoma was recently classified by a large genomic study into four molecular subtypes, including one defined by Epstein-Barr virus (EBV)-positivity. EBV infection in tumors is conventionally assessed by in situ hybridization (ISH), but detection of viral nucleic acids by next-generation sequencing represents a potential alternative. Methods: Normalized EBV read counts were determined by whole genome, whole exome, mRNA and miRNA sequencing for 295 fresh-frozen gastric tumor samples. Formalin-fixed, paraffin-embedded tissue sections were retrieved for ISH confirmation of 13 high-EBV and 11 low-EBV cases selected at random. Results: By each sequencing method, numbers of EBV reads were bimodally distributed across tumors, with a minority having much higher counts. In pairwise comparisons, individual samples were either consistently high or consistently low by all genomic methods for which data were available. Empiric cut-offs based on molecular data of 1000 normalized reads for whole genome, 100 for exome, 4 for mRNA and 5000 for miRNA had perfect concordance identifying 26 (9%) tumors as EBV-positive. One sample tested by ISH lacked tumor cells. For the 23 tumors with EBV status determined by both approaches, EBV-positivity or -negativity by molecular testing was confirmed by EBER-ISH in all but one case (kappa = 0.91). The exception was a microsatellite instability-type cancer EBV-negative by both mRNA and miRNA sequencing, with equivocally positive ISH interpretation. Conclusions: EBV-positive gastric tumors may be accurately identified by quantifying viral sequences in genomic data. Simultaneous analyses of human and viral DNA, mRNA and miRNA could streamline tumor profiling for clinical care and research. Citation Format: M. Constanza Camargo, Reanne Bowlby, Andy Chu, Chandra S. Pedamallu, Vesteinn Thorsson, Sandra Elmore, Andrew Mungall, Adam Bass, Margaret L. Gulley, Charles S. Rabkin. Validation and calibration of next-generation sequencing to identify Epstein-Barr Virus-positive gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4621. doi:10.1158/1538-7445.AM2015-4621